The objective of this core TR&D project is to test, develop and improve additional selective stains for cellular and subcellular studies with special emphasis on methods involving photo-oxidation for correlative light and electron microscopy and to determine the feasibility of extending this methodology to dyes for capturing dynamic changes in subcellular chemistry at high resolution. The most significant progress in this area has been in the use of photooxidation of eosin for selective staining. At the end of last year, we had determined that eosin was an excellent fluorophore for correlated light and electron microscopic data on subcellular structures. Although we primarily have been using eosin for immunolocalization, based on our results, several companies have been offering eosin-conjugated reagents such as eosin-phalloidin, eosin-bungarotxin and eosin-WGA for nonimmunocytochemical staining. We have obtained excellent results staining and photoconverting actin with phalloidin, the neuromuscular junction with bungarotoxin and the muscle T-tubules with WGA. These preparations have all been used for correlated light and electron microscopic analysis. In addition to the work with eosin, we have been using additional fluorescent organelle-specific stains for photo-oxidation. These include propidium iodide to stain the chromosomes in mitotic cells and Syto dye, which stains ribosomes. The Syto dye work was performed as part of a collaboration between Dr. Ken Kosik and Roger Knowles to investigate the transport of RNA into the dendrites of developing nearites. This project is presented in some detail in a later section. We will begin tests to determine the feasibility of photo-converting the green fluorescent protein (OFP) so that individual proteins can be visualized in living cells, photoconverted and examined under the electron microscope. Some groups have reportedly been successful in photo-converting UFP. A third area of progress in this core area involves the use of tomography and selective stains to reconstruct organelles. Guy Perkins, a post-doctoral researcher in the laboratory, is using this approach to study the structure of mitochondrial cristae in axons and dendrites.